PROJECT SUMMARY: There has been no improvement in the cure rate for epithelial ovarian cancer (EOC) for the past 30 years. Approximately 21,400 women in the U.S. are diagnosed annually, and ~15,000 will develop fatal chemoresistant disease. Thus, overcoming chemoresistance is a critical unmet need. One target to overcome chemoresistance is the ALDH1A family of isoenzymes. ALDH1A isoenzymes are upregulated in chemoresistant ovarian cancer cells in vitro and in vivo, and knockdown of ALDH1A1 reverses chemotherapy resistance. Potentially related to its role in chemotherapy resistance, highly ALDH-active (ALDHbright) cells can act as cancer stem-like cells (CSC). ALDHbright ovarian CSC are chemoresistant with increased tumor initiation capacity. Experimentally, agents targeting CSC can increase chemotherapeutic efficacy, reverse chemo- therapy resistance, and prevent cancer recurrences. Thus, targeting ALDH1A isoenzymes may have the unique benefit of targeting CSC. Indeed, our preliminary data suggests that a novel ALDH1A inhibitor 673A (i) synergizes with chemotherapy to kill chemoresistant ovarian cancer cells, (ii) selectively kills ovarian CSC, thereby reducing tumor initiation capacity, and (iii) in multiple tumor models combined with chemotherapy is able to cure ~50% of animals with chemoresistant ovarian cancer. Unfortunately, 673A and other analogs are poor candidates for clinical development as they are rapidly metabolized and/or are covalent ALDH modifiers. We have recently identified a novel series of potent non-covalent, non-substrate ALDH1A inhibitors. Importantly, our preliminary data demonstrate that these new ALDHi kill CSC and synergize with chemotherapy. Isoenzyme selectivity of the ALDHi can be readily modified by minor structural changes. The overarching goal of this work will be to determine if isoenzyme-selective inhibition of ALDH1A will constitute a safe and effective adjunct for sensitizing the treatment of recurrent ovarian cancer to existing chemotherapy. We will also identify which ALDH1A isozyme inhibition profile(s) is/are most effective. We propose to: 1) optimize ALDH1A inhibitor based on our novel chemical series for potency and isozyme selectivity, guided by new co-crystal structural information; 2) determine which ALDH1A inhibitors most effectively deplete ovarian CSC in vitro; 3) evaluate optimal inhibitors for pharmacokinetic properties; and 4) select optimal probes for evaluation in murine xenograft models of chemoresistant ovarian cancer. Our proposal is innovative in its use of a new ALDH1A : inhibitor co-crystal structure and homology models for structure-based design of isozyme- selective inhibitors, and the ultimate use of patient-derived xenografts with human stroma to more accurately predict efficacy in the clinic. This work will be significant in determining whether ALDH1A family inhibition can overcome ovarian cancer chemoresistance in vivo and which isozyme inhibition profile is optimal. Finally, our work will progress toward the development of an effective adjunct treatment for recurrent ovarian cancer, a dire unmet medical need, thus there is high potential for significant impact.